BE115+-+Lecture06 - Lecture 6 SDS-PAGE "Trying to determine...

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Lecture 6 SDS-PAGE “Trying to determine the structure of a protein by UV spectroscopy was like trying to determine the structure of a piano by listening to the sound it made while being dropped down a flight of stairs.” - Francis Crick 1
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Path to Protein Analysis Cell culture, tissue culture, or other experiment Culture or tissue extraction Sample preparation Protein purification Total protein quantification Analysis Given a particular protein of interest, what might I want to know about it? 2
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Questions for Protein Analysis Which proteins are in my sample? What is the condition of the proteins? Are they whole? Are they active? Are they associated with other proteins? Did my experimental conditions change the amount or condition of a particular protein? 3
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Protein Characteristics Mass Size Proteins of the same mass can have different shapes depending on their secondary and tertiary structure Hydrophobicity Antigenicity Activity Phosphorylation state Etc. 4
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History Electrophoresis was pioneered by Arne Tiselius He was awarded the 1948 Nobel prize in chemistry for his work 5 Tiselius’ original electrophoresis apparatus
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Electrophoresis Separates molecules in a complex mixture by size and charge Uses electrical field through a gel with regular/known pore size Proteins typically in acrylamide gels Nucleic acids typically in agarose gels “Charge” can be native to the protein or added during the experiment 6
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SDS-PAGE Electrophoresis separates molecules in a complex mixture by both size and charge SDS-PAGE separates proteins by size How can we ignore protein charge in SDS- PAGE? 7
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SDS Detergent that associates with proteins (~1.4g SDS/g protein) Protein charge is small compared to cloud of negatively charged SDS A protein gets a negative charge roughly proportional to its mass Reducing agents (like b-mercaptoethanol and DTT) are often used to reduce disulfide bonds and further eliminate secondary structure 8 Hydrophobic regions SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS SDS
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2X Sample Buffer Added to samples before they are loaded into the gel Contains: SDS β-mercaptoethanol (reducing agent) Tris (buffer) bromophenol blue (dye) glycerol (increases sample density) 9
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SDS-PAGE S odium D odecyl S ulfide P oly A crylamide G el E lectrophoresis 10
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Gel components Acrylamide (polyacrylamide) Crosslinker (N’N’bis-methylene- acrylamide or “bis”) Catalyst (TEMED) Initiator (ammonium persulfide or “APS”) Buffer (Tris-glycine) Water 11
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Crosslinking Acrylamide alone makes long polymer segments (end- on-end) Bis-acrylamide acts as a crosslinker More bis = more crosslinks = stiffer gel = smaller pores 12 TEM of a polyacrylamide gel
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Gel pore size 29:1 acrylamide:bis-acrylamide ratio for proteins 19:1 acrylamide:bis-acrylamide ratio for
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This note was uploaded on 10/14/2011 for the course BIOE 115 taught by Professor Haley during the Spring '11 term at City College of San Francisco.

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BE115+-+Lecture06 - Lecture 6 SDS-PAGE "Trying to determine...

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